The basic features of the cortical cytoskeleton of the red cell are now understood in considerable detail. Nearly a score of inherited diseases with erythrocyte instability have been linked to specific molecular defects in one or the other of its major proteins. However, our understanding of the role of other erythrocyte cytoskeletal proteins, or even how many additional proteins there are that interact with the spectrin skeleton, how they are regulated by post-translational modification or allosteric mechanisms, how they participate in signal transduction events or in other cellular processes, even what diseases result from their dysfunction, remains rudimentary. In addition, the structural basis of most interactions between spectrin and ankyrin and other ligands is completely unknown (beyond their primary sequence). Building on our premise that the spectrin/ankyrin cytoskeleton bestows stability on the plasma membrane by controlling membrane protein and possibly even lipid organization, both during erythrocyte maturation and in the mature cell, and serves as a signal scaffold for several regulatory pathways, the proposed studies continue our long-term focus on spectrin as the central component of the cytoskeleton, and our goal of fully understanding the molecular basis of erythrocyte membrane organization, dynamics, cooperative regulation, and dysfunction. Two complimentary specific approaches will be pursued in the proposed studies: 1) identification of novel erythrocyte proteins that interact with spectrin or ankyrin, delineation of their sites of interaction within spectrin and/or ankyrin using high-throughput screens based on in-vitro binding assays and genetic selection;and their purification and validation via quantitative in vitro assays;and 2) Resolution of the 3-D structure of protein-protein complexes critical for spectrin-ankyrin function by X-ray crystallography. The complexes of immediate interest include the two-repeat ankyrin-binding domain of spectrin complexed with the spectrin-binding domain of ankyrin;the structure of the paired ?l/?l repeats implicated in the allosteric control of spectrin's self-association;and the structure of the paired ?l/?I sequences responsible for nucleating heterodimer assembly. These studies will enhance our understanding of the spectrin cytoskeleton, identify the structural basis underlying the pathology of certain inherited diseases, and extend the generality and significance of the erythrocyte paradigm for the study of more complex cells.